The authors would like to thank Prof. Steven Nutt and Mr. Yuzheng Zhang for assistant with the Focus Ion Beam, and the Center for Electron Microscopy and Microanalysis (CEMMA) at USC for electron microscopy. Research was supported by NIH-NIDCR grants; DE-13414 and DE-020099 to J.M.O.

人間の臼歯は、南カリフォルニア大学の歯学部のオストロー学校での抽出のための標準的な手順に従って抽出し、施設内倫理委員会の承認を得て取り扱った。 1。酸エッチング歯の作製スライス<ol><li>任意の復元された虫歯なしで、人間の第三大臼歯を選択します。 </li><li>臼歯の根元部分を削除し、縦方向に太い水冷式​​低速ダイヤモンドソーを用いてスライス2ミリメートルに臼歯の冠をカット。超音波で2分間、これらのスライスをきれいにした後、脱イオン水で3回、それらをすすぐ。 </li><li>びらん性病変をシミュレートするために、直ちに脱イオン水ですすぎ、その後、それらを30秒間、30％リン酸で歯スライスをエッチングする。 </li><li>超音波で2分間エッチングしたスライスをきれいにした後、脱イオン水で3回、それらをすすぐ。 </li></ol>アメロゲニン - キトサンハイドロゲルの2。準備<ol><li> Preparatioキトサン原液のN <ol><li> 1％を溶解し、80℃CO / Nで攪拌し、続いて1％（v / v）酢酸溶液（w / v）のキトサン（中間分子量、75〜85％脱アセチル化） </li><li> RTに冷却後、溶液を、0.45μmのフィルターを用いてキトサン溶液を濾過する。 1 M NaOH溶液を添加することにより5.0にpH値を調整する。 </li></ol></li><li>カルシウム及びリン酸のストック溶液（0.1 M）の調製<ol><li>塩化カルシウム（CaCl 2で） を秤量し、0.1 M溶液を調製し、次いで、溶液が透明になるまでボルテックスを用いて混合する。 1 M NaOH溶液を加えることにより11にpH値を調整する。 </li><li>第二リン酸ナトリウムせ（Na 2 HPO 4）を秤量し、0.1 M溶液を調製し、次いで、溶液が透明になるまでボルテックスを用いて混合する。 </li></ol></li><li>組換えアメロゲニン17〜18の発現および精製 ここで使用される組換え完全長ブタアメロジェニン（rP172）は、172アミノ酸を有し、完全にアナログである長天然ブタP173が、N末端 ​​メチオニンなどSer16 18上のリン酸基を欠いている。 <ol><li> 500mlの溶原性ブロス（LB）寒天の粉末20gを追加し、脱イオン水、20分間121°C（液体設定）で溶液をオートクレーブ。 〜55°Cは、寒天液にアンピシリン（100μg/ mlの）を追加する寒天クールをしてみましょう。 </li><li>寒天の結露を避けるためにフリップして、それが固体になるまで、各プレートが冷めて、ペトリ皿に寒天ソリューションを転送します。 4℃で、ビニール袋に入れて保管してプレート</li><li>グリセロールストックからの組換え細胞（ 大腸菌 -BC21）でストリークLB寒天プレート（-80℃）。 37℃でプレートO / Nインキュベート</li><li> NZCYM培地を50mlを調製し、30分間121℃でオートクレーブ媒体。メディアがクールダウンするには、50ミリリットルのメディアに100μg/ mlのアンピシリンの50μlを加える。 </li><li> ampicillを補充し、50mlのNZCYM培地にLB寒天プレートからの単一コロニーを接種インチ、37℃で振とう培養器内の細胞のO / Nインキュベート</li><li> NZCYMメディアを1リットル調製し、30分間121℃でメディアをオートクレーブ。千ミリリットルメディアに100 mg / mlのアンピシリンの1ミリリットルを追加します。 UV-可視分光光度計を用いてブランクとして光学密度（OD）読み取り値を取る。注：最初にリードした後は捨てないでください。 </li><li>ステップ2.3.6から千ミリリットルメディアにステップ2.3.5からの細胞培養の10ミリリットルを接種する。すぐに接種後595nmでの光学密度を読んでください。成長を追跡するために、定期的に光学濃度の読みを取る。注：外径が取られるたびに空白をお読みください。 </li><li>細菌の増殖は、595nmで約0.75〜0.8 ODまで到達したときにイソプロピルβ-D-1-チオガラクトピラノシド（IPTG、1 M）の700μlで誘導する。 </li><li> ODが1.1に達したときに細胞を採取する。 6プラスチックボトルにフラスコの内容物を注ぐ。バランスをとり、遠心分離器を使用する前に、ボトルの重量を量る。 4℃で8554×gで20分間遠心-20°CO / Nで細胞ペレットを保つ</li><li>細胞ペレットを取り出し、50mlチューブでそれらを結合します。発酵の1Lあたり4MグアニジンHClの3.5ミリリットルを追加します。 1分間隔で超音波処理し、氷上に2〜3回、30秒ごとに、。 </li><li> 0.5％ギ酸で細胞容積の6倍希釈液を作り、それが2時間低温室で炒めてみましょう。 4℃で30分、8554×gで遠心上清を保管してください。飽和の原液を用いて20％飽和硫酸アンモニウムを加え、低温室、O / Nでかき混ぜる</li><li> 4℃で30分間、8554×gで遠心し、ペレットを保持します。 C4カラム（10×250ミリメートル、5ミクロン）上に0.1％トリフルオロ酢酸（TFA）、負荷のペレットを再構成し、そしてにおいて、TFAでA = 0.1％TFAおよびB = 60％アセトニトリルの直線勾配を用いて分画し1.5ミリリットル·分-1の流速。 </li><li>ドライアイス上で蛋白質溶液を凍結し、凍結したサンプルのO / Nを凍結乾燥</li></ol></li><li>アメロゲニン - キトサンの調製（CS-AMEL）hydrogEL（ 図1A） <ol><li> 200 rp172μgの1％キトサン溶液を960μLを含むチューブに追加し、溶液が透明になるまでボルテックスを用いて混合します。 </li><li>アメロゲニン含有キトサン溶液に、0.1MのNa 2 HPO 4溶液を15μlに続く、0.1MのCaCl 2溶液25μlを追加し、5分間ボルテックスを用いて混合します。 </li><li>慎重に1 M NaOH溶液を添加することによって6.5にCS-AMEL溶液のpHを調整する。 pH値が6.5に達したときにCS-AMELヒドロゲルが形成される。 </li></ol></li></ol>アメロゲニン·キトサンヒドロゲル中の3。エナメル再成長<ol><li>人工唾液溶液の調製<ol><li>特定の塩化マグネシウム（MgCl 2を 、0.2 mM）の量は、塩化カルシウム（CaCl 2、1mM）を、リン酸二水素カリウム（KH 2 PO 4、4ミリモル）、塩化カリウム（KCl、16 mM）および塩化アンモニウム（溶解20のHEPES中のNH 4 Clで、4.5 mM）を（4 - （2 -ヒドロキシエチル）ピペラジン-1 -エタンスルホン酸）緩衝液12。 </li><li> 1MのNaOHでpHを7.0に調整し、4℃で、人工唾液ソリューションを保存する</li><li>ソリューションを使用する前に、フッ化ナトリウム（NaFを、300 ppm）を追加します。 </li></ol></li><li> CS-AMELハイドロゲルの応用<ol><li>注射器（ 図1B）を用いてエッチング歯のスライスに約20μlのCS-AMELハイドロゲルを適用します。 </li><li>室温で2時間デシケーター中でハイドロゲルで覆わ​​れた歯のスライスを乾燥させます。 </li><li>人工唾液溶液30mlを入れたビーカーに歯のスライスを転送します。アルミニウム箔でビーカーを覆い、その後、7日間37℃のオーブン中にビーカーを保つ。 </li><li>歯のスライスを削除して、室温でデシケーター中で乾燥させます。 </li></ol></li></ol>新成長層とエナメルとの間のインタフェースの4。キャラ新しく成長した層とエナメルの界面の微細構造はobserた電子顕微鏡（SEM）及び高回転透過型電子顕微鏡（HR-TEM）を走査することによってVED。 TEM試料は、集束イオンビーム（FIB）技術、次の通りであるれる手順を用いて調製した。 <ol><li> FIB-SEM装置にサンプルをロードし、取扱説明書に従ってすべての整列を終える。 NOTE：微調整Zは、少なくとも2回実施されるべきである。 </li><li>基本的な構造（ 図2A）を保護するために試料上にカーボン層（15×3程度）を蒸 ​​着した。 </li><li>ミルは、炭素層の上部、下部、右両側に注意深く試料は、試料の薄片を作製し、この薄片の底部側をカットする。注：各粉砕工程（ 図2B）の前にイオンビームの位置合わせを確認してください。 </li><li>溶接白金薄片上のチップと試験片（ 図3C）を分離するために左側を切った。注：溶接工程の前にイオンビームの位置合わせを確認してください。 </li><li> Tを持ち上げ彼は徐々にサンプルとヒントリフトアウトTEMグリッド上にラメラサンプルをマウントPT。 </li><li>その厚さが100nm（ 図3D）未満になるまで、サンプルから先端を切り離し、薄いサンプル。 TEM観察を行うために、機器からサンプルを取り出します。注：各間引き処理の前にイオンビームの位置合わせを確認してください。 </li></ol> 5。新成長層の結合および機械的特性の評価を<ol><li>結合強度は、超音波処理することにより評価される。 10分間の超音波洗浄機（42 kHzの、100 W）で修復歯のスライスを維持した後、後方散乱電子SEM分析と修復さ層と天然エナメル質との間のインタフェースを観察します。 </li><li>ベルコビッチチップを有するナノインデンターにより各補修エナメル質表面上に20のテストポイントでの硬度及び弾性率を測定した（n = 3）。 </li></ol> 6。免疫蛍光染色<ol><li>トリスbuffeと歯のスライスを洗う15分間のr個の生理食塩水（TBS）。 </li><li> 15分間、TBS中の1％ウシ血清アルブミン（BSA）でブロックする。 </li><li>液体を除去し、TBS（0.1％のBSA、0.3％トリトンX-100）で希釈した1°抗体（1:500ニワトリ抗アメロゲニン）とサンプルのO / Nインキュベートする。 </li><li> 30分間TBSでサンプルを洗浄し、TBSで希釈した2°抗体（1:100抗ニワトリFITC）でO / Nインキュベートする。 </li><li> 30分間TBSでサンプルを洗浄し、さらに30分間乾燥さのままにしておきます。 </li><li>蛍光顕微鏡で観察します。 </li></ol>

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The authors have nothing to disclose.

エナメル質のミネラル含有量が高く、それを人間の体内で最も困難なミネラル化した組織を作っている間に、このバイオセラミックは、多くの場合、虫歯やエロージョンとして生じる脱塩プロセスの影響を受けやすい。遺伝子突然変異はまた、 エナメル質形成不全症20と呼ばれるエナメル奇形の遺伝性疾患の一連につながる細いまたはソフトエナメルを引き起こす可能性があ?...

Dental enamel is the hard mineralized surface of human teeth. It is composed of numerous needle-like apatite crystals, which are bundled in organized, parallel prisms to ensure the unique mechanical strength and biological protection that enamel provides1-2. Unlike other mineralized tissues, such as bone and dentin, mature enamel is acellular and cannot regenerate itself after substantial mineral loss1-2, which often occurs as dental caries or erosion. Commercially available products such as fluoride containing varnishes, tooth pastes and mouthwashes are effective in re-mineralizing enamel but none of them have the potential to promote the formation of organized apatite crystals. Clinically, the conventional treatment for enamel repair involves a filling procedure with artificial materials such as amalgam, ceramics, or composite resin 3. However, these materials usually do not interface well with the natural tissue surrounding the lesion, because their structures, components and properties are different from the natural enamel. As a result, secondary caries frequently develops overtime at the interface between the tooth and foreign materials. Therefore, in situ regrowth of enamel with a dense interface is an attractive target for the fields of materials science and stomatology. One particularly promising way to achieve this purpose is biomimetic synthesis of enamel-like material on the enamel surface. Recently, numerous in vitro attempts have been made to prepare enamel-like materials using biomimetic systems that contain nano-apatites or different organic additives 4-12. However, developing the optimal biomimetic strategy to promote remineralization crystals to achieve a perfect dense interface is still a challenge. 
During enamel mineralization, the oriented growth and elongation of apatite crystals is regulated by an amelogenin-rich matrix 1,13-14. To mimic the organic matrix in developing enamel, herein we describe a detailed protocol for fabricating an amelogenin-chitosan (CS-AMEL) hydrogel for in situ enamel regrowth on an acid-etched enamel surface used as a model for erosive lesions. As &#8220;the most versatile growth media&#8221; for crystals 15, hydrogel matrices have an advantage over a solution system since clinically they are easier to handle. Moreover, CS-AMEL is biocompatible, biodegradable, and has unique antimicrobial and adhesion properties that compare favorably with other biomimetic systems for dental applications 16. Importantly, the in situ mineralization of apatite crystals on the enamel surface provides a dense interface between the repaired layer and the natural enamel, which can potentially improve the durability of restorations and prevent the formation of new caries at the margin of the restoration.

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			<td height="21" style="height:21px;width:331px;">Name</td>
			<td style="width:209px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:516px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Material/ Reagent</td>
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			<td style="width:164px;"></td>
			<td></td>
		</tr>
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			<td height="63" style="height:63px;width:331px;">Human Third Molar&#160;</td>
			<td style="width:209px;">Ostrow School of Dentistry of the University of Southern California&#160;</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">The human molars were extracted following the standard procedures for extraction at the Ostrow School of Dentistry of the University of Southern California and handled with the approval of the Institutional Review Board.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:331px;">Recombinant Pocine Amelogenin</td>
			<td style="width:209px;">Expression and purification&#160; in lab</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">rP172, full-length&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chitosan&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">448877</td>
			<td style="width:516px;">medium molecular weight, 75-85% deacetylated</td>
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			<td height="21" style="height:21px;width:331px;">Phosphoric Acid&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA033266</td>
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			<td height="21" style="height:21px;width:331px;">Acetic Acid Glacial</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">A036289</td>
			<td style="width:516px;"></td>
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			<td height="21" style="height:21px;width:331px;">Sodium Hydroxide</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9292</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Calcium Chloride&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">223506</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Dibasic Sodium Phosphate Anhydrous</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0316</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">BL21-CodonPlus (DE3)-RP&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">230255</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Sulfate</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH8001</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Trifluoroacetic Acid</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAL06374</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Acetonitrile</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH1103</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Magnesium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0244</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Dihydrogen Phosphate&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9268</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0258</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAA15000</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid)</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAA14777</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Fluoride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA11561</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tris-Buffered Saline</td>
			<td style="width:209px;">Bio-Rad</td>
			<td style="width:164px;">170-6435</td>
			<td style="width:516px;">10&#215; TBS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Serum Albumin</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">12659</td>
			<td style="width:516px;">CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Triton X-100</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">TX1568-1</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chicken Anti-Amelogenin</td>
			<td style="width:209px;">N/A</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">A gift from Prof. Malcolm Snead, University of Southern California</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Anti-Chicken IgY-FITC</td>
			<td style="width:209px;">Santa Cruz Biotechnology</td>
			<td style="width:164px;">Sc-2700</td>
			<td style="width:516px;"></td>
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			<td height="32" style="height:32px;width:331px;">Equipments</td>
			<td style="width:209px;"></td>
			<td style="width:164px;"></td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:331px;">High Performance Liquid Chromatography System</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">Varian Prostar 210</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">C4 column</td>
			<td style="width:209px;">Phenomenx&#160;</td>
			<td style="width:164px;">Jupiter 5&#956; 300A</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Scanning Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JSM-7001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FIB-SEM&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JIB-4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Transmission Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL</td>
			<td style="width:164px;">JEM-2100F</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital Low Speed Diamond Saw</td>
			<td style="width:209px;">MTI Corporation</td>
			<td style="width:164px;">SYJ-150</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fluorescence Microscopy</td>
			<td style="width:209px;">Leica</td>
			<td style="width:164px;">DMI3000 B</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ultrasonic Cleaner&#160;</td>
			<td style="width:209px;">Branson&#160;</td>
			<td style="width:164px;">2510</td>
			<td style="width:516px;">42 kHz, 100 W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nano-indenter&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">MTS XP</td>
			<td style="width:516px;"></td>
		</tr>
	</tbody>
</table>

development of amelogenin-chitosan hydrogel for in vitro enamel regrowth with a dense interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

ここに記載されたプロトコルの有効性は、電子顕微鏡（SEM）、選択領域の電子回折（SAED）及びX線回折（XRD）分析を走査することによって実証される。 7日間アメロゲニン - キトサン（CS-AMEL）ハイドロゲルによる修理の後、厚さ15μmのエナメル調層がエッチングされたエナメル質表面に形成された。新たに成長した層は、表面に垂直なc軸（ 図3Aの矢印）に沿って優先的に成長し、...

Watch this Scientific Journal Video about Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface at JoVE.com

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

この記事では、我々は表面的なエナメル質の再構築のためのアメロゲニン-キトサンハイドロゲルを製造するためのプロトコルを記述します。ヒドロゲル中のアパタイト結晶のその場での増殖に編成は、修復物の有効性及び耐久性を向上させる緻密なエナメル復元インタフェースを形成した。

用アメロゲニン - キトサンハイドロゲルの開発<em&gt;体外</em高密度インターフェイス付き&gt;エナメル再成長

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

development-amelogenin-chitosan-hydrogel-for-vitro-enamel-regrowth

Research

JoVE Journal

Bioengineering

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

14.9K ビュー.  University of Southern California. この記事では、我々は表面的なエナメル質の再構築のためのアメロゲニン-キトサンハイドロゲルを製造するためのプロトコルを記述します。ヒドロゲル中のアパタイト結晶のその場での増殖に編成は、修復物の有効性及び耐久性を向上させる緻密なエナメル復元インタフェースを形成した。 

ビデオ: Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心臓組織工学のためのフィブリンゲルにおける心筋細胞のカプセル化

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

生物工学

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3次元自己組織化ペプチドハイドロゲル中のヒト神経前駆細胞の栽培

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Biofabricationとバイオエレクトロニック·インタフェースをブリッジする

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

インテイン媒介人工タンパク質ヒドロゲルの合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

ハイドロゲルナノ粒子プラズマの収穫や少量のタンパク質を検出するための尿

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

血管細胞リモデリングの研究への応用生理学的および病理学的血流のインビトロモデル

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

開発<em&gt;インビトロ</em&gt;眼プラットフォームはコンタクトレンズをテストします

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

プライマリのグリア細胞の in Vitro Neuroinflammation のモデリングのための三次元ハイドロゲル文化を改善

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable アルギン酸/ゼラチン ゲル 3 Dの in Vitroモデル システム細胞スフェロイドの形成を誘導します。

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...